RESUMEN
BACKGROUND: More than a dozen of fungal immunomodulatory proteins (FIPs) have been identified to date, most of which are from Ganoderma species. However, little is known about the similarities and differences between different Ganoderma FIPs' bioactivities. In the current study, two FIP genes termed FIP-gap1 and FIP-gap2 from G. applanatum, along with LZ-8 and FIP-gsi, another two representative Ganoderma FIP genes from G. lucidum and G. sinense were functionally expressed in Pichia. Subsequently, bioactivities of four recombinant Ganoderma FIPs were demonstrated and compared. RESULTS: All the four Ganoderma FIP genes could be effectively expressed in P. pastoris GS115 at expression levels ranging from 197.5 to 264.3 mg L- 1 and simply purified by one step chromatography using HisTrap™ FF prepack columns. Amino acid sequence analysis showed that they all possessed the FIP conserved fragments. The homologies of different Ganoderma FIPs were from 72.6 to 86.4%. In vitro haemagglutination exhibited that FIP-gap1, FIP-gsi and LZ-8 could agglutinate human, sheep and mouse red blood cells but FIP-gap2 agglutinated none. Besides, the immunomodulation activities of these Ganoderma FIPs were as: rFIP-gap2 > rFIP-gap1 > rLZ-8 and rFIP-gsi in terms of proliferation stimulation and cytokine induction on murine splenocytes. Additionally, the cytotoxic activity of different FIPs was: rFIP-gap1 > rLZ-8 > rFIP-gsi > rFIP-gap2, examined by their inhibition of three human carcinomas A549, Hela and MCF-7. CONCLUSIONS: Taken together, four typical Ganoderma FIP genes could be functionally expressed in P. pastoris, which might supply as feasible efficient resources for further study and application. Both similarities and differences were indeed observed between Ganoderma FIPs in their amino acid sequences and bioactivities. Comprehensively, rFIP-gaps from G. applanatum proved to be more effective in immunomodulation and cytotoxic assays in vitro than rLZ-8 (G. lucidum) and rFIP-gsi (G. sinense).
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/farmacología , Ganoderma/genética , Expresión Génica , Factores Inmunológicos/genética , Factores Inmunológicos/farmacología , Secuencias de Aminoácidos , Animales , Línea Celular , Citocinas/genética , Citocinas/inmunología , Eritrocitos/efectos de los fármacos , Eritrocitos/fisiología , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Ganoderma/química , Ganoderma/metabolismo , Pruebas de Hemaglutinación , Humanos , Factores Inmunológicos/aislamiento & purificación , Factores Inmunológicos/metabolismo , Ratones , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , OvinosRESUMEN
Fungal immunomodulatory proteins (FIPs) have been identified from a series of fungi, especially in Ganoderma species. However, little is known about the FIPs from G. applanatum. In this study, two novel FIP genes, termed as FIP-gap1 and FIP-gap2, were cloned from G. applanatum, characterized and functionally expressed after codon optimization in Pichia pastoris GS115. Results showed that FIP-gap1 and FIP-gap2 comprised 342-bp encoding peptides of 113 amino acids, which shared a high homology with other Ganoderma FIPs. The yield of recombinant FIP-gap1 and FIP-gap2 increased significantly after codon optimization and reached 247.4 and 197.5 mg/L, respectively. Bioactivity assay in vitro revealed that both rFIP-gap1 and rFIP-gap2 could agglutinate mouse, sheep, and human red blood cells. Besides, rFIP-gap1 and rFIP-gap2 obviously stimulated the proliferation of mouse splenocytes and enhanced IL-2 and IFN-γ release. Cytotoxicity detection indicated that IC50 of rFIP-gap1 towards A549 and HeLa cancer cells were 29.89 and 8.34 µg/mL, respectively, whereas IC50 of rFIP-gap2 to the same cancer cells were 60.92 and 41.05 µg/mL, respectively. Taken together, novel FIP gaps were cloned and functionally expressed in P. pastoris, which can serve as feasible and stable resources of rFIP gaps for further studies and potential applications.
